Fowl cholera: Evaluation of a Trivalent Pasteurella multocida Vaccine Consisted of Serotypes 1, 3 and 4

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1 Arch. Razi Ins. 59 (2005) Fowl cholera: Evaluation of a Trivalent Pasteurella multocida Vaccine Consisted of Serotypes 1, 3 and 4 Short Communication Jabbri *, A. R., Moazeni Jula, G.R. Aerobic Veterinary Bacterial Vaccines Research & Production Dept., Razi Vaccine & Serum Research Institute, P.O.Box , Tehran, Iran Received 23 Dec 2004; accepted 29 Mar 2005 Summary An inactivated trivalent fowl cholera vaccine consisted of serotypes 1, 3 and 4 Pasteurella multocida strains was prepared. The vaccine provided % protection against challenge with homologous starins. ELISA assay showed a considerable increase in antibody titer after towice vaccination of 8 weeks chickens. It was found that the trivalent vaccine can induce immunogenic response in vaccinated chickens. Key words: Pasteurella multocida, fowl cholera, trivalent vaccine, ELISA Introduction Fowl cholera, caused by P.multocida can result in either an acute septicemia or chronic localized infections in domestic and wild birds (Sander et al 1998). Among the bacterial diseases of domestic birds, fowl cholera accounts for major economic losses to the industry through death, weight loss and condemnations. The disease has been recognized for over 200 years and has been the subject of many researches. Despite this attention fowl cholera still remains a problem in the modern poultry industry (Rimler & Roades 1989) In Iran, since the first report of fowl cholera reported in 1971 (Bozorgmehrifard & Afghan) outbreaks of the disease have been reported by the Veterinary Organization in the northern part of Iran where fowl cholera is endemic (Kalaydari et al 2004, * Author for correspondence. ahmadjb@yahoo.com

2 104 Jabbari & Moazeni Jula/Arch. Razi Ins. 59 (2005) Tavasoli et al 1984). A monovalent killed aluminum hydroxide vaccine prepared from serotype A1 P.multocida (Sotoodehnia et al 1986) in Razi Vaccine & Serum Research Institute, is used for induction of immunity against avian cholera. It was demonstrated that the killed vaccine protected the chickens against homologous challenge (Sotoodehnia et al 1984) however; in spite of vaccination in the endemic area outbreak of fowl cholera has been reported (Kalaydari 1998). Bacterins have been widely used to prevent fowl cholera however these vaccines generally afforded homologous but not heterologous protection (Petersen et al 1991). Recently, presence of other serotypes of P.multocida was reported from Iran (Jabbari et al 2001). This investigation was undertaken to develop a trivalent vaccine containing serotypes 1, 3 and 4 of P.multocida strains and to evaluate its efficacy in chickens. Materials and Methods Chickens and laboratory animals. 8-week-old chickens were raised in an isolated facility. Feed and drinking water were available all the time. The mouse and rabbits for pathogenecity study were obtained from Laboratory Animals Productions Department, Razi Institute. Vaccine preparation. The freeze dried stocks of serotype 1 (PMI030 strain), serotype 3 (PMI035 strain) and serotype 4 (PMI047 strain) of P.multicided isolated from field and natural outbreaks of fowl cholera in the northen provinces were used. Phenotypic and molecular characterization, and the minimum lethal dose (MLD) as a virulence indicator of the isolates were described previously (Jabbari et al 2002a, b, 2003). After reconstitution with 0.5ml triptose phosphate broth (TPB), each strain was streaked on sheep blood agar plate and incubated for 24h at 37 C. A single typical colony was cultured in 300ml TPB and incubated for 18h at 37 C with shaking. Cell suspensions was inactivated by adding 0.3% formaldehide and was left to stand for 24h. The cells were separated by centrifugation at 5000g for 30min and the pellet was resuspended in PBS (ph7.2). The optical density of each suspension

3 Arch. Razi Ins. 59 (2005) was adjusted to 1 spectrophotometerically (Ultraspec 2000, Pharmacia) at 540nm. The aluminum hydroxide gel, as an adjuvant, was added to a mix equal proportion of each strain up to 1% final concentration. The vaccine was stored at 4 C. The purity and sterility tests were done according to OIE manual (2001). Immunization and challenge procedure. 8-week-old SPF chickens were divided in two vaccinated and control groups. 1 ml dose of the prepared vaccine was intramascularly administrated and vaccination was repeated 3 weeks later. The chickens were challenged 2 weeks after booster injection. To challenge a typical colony of each strain was transferred to 10ml TPB and incubated for 6h at 37 C. The optical density of these cultures was then adjusted spectrophotometrically to at 540nm. The concentration of these cultures was approximately CFU/ml. The cultures were diluted serially ten-fold in PBS and selected dilutions were used for challenging. For estimating the concentration of viable organisms, 0.5ml of selected suspensions were spread on BA plates. The colonies were counted after 24h incubation at 37 C. Blood samples were taken from the wing web brachial vein of all chickens before first vaccination, booster and before challenge exposure. Sera were collected and stored at 20 C until use. Serological assay. An ELISA test was used to evaluate the immune response of vaccinated chickens by using two antigens, whole cell and sonicated antigens. Whole cell antigen was prepared according to the Kedrak et al (2000) method with some modifications. Briefly, the antigen was prepared using 18-h culture of agitated P.multocida in TPB medium at 37 C. The culture was inactivated with 0.3% formalin, centrifuged twice and washed with PBS, ph7.2. The bacterial pellet was suspended in carbonate buffer, ph9.6. The suspension was adjusted to optical density of at 540nm spectrophotometrically. Antigens were stored at 20 C until use. In prelimnary examination optimum dilution of whole cell antigen was evaluated as 1:10. Sonicated antigen was prepared as described by Perelman et al. (1990). Coating and blocking of microplate (Polysorb, Nunc) and ELISA procedure

4 106 Jabbari & Moazeni Jula/Arch. Razi Ins. 59 (2005) were carried out according to Avakian and Pick (1985). Absorbancy of the plates were read at 405nm by an ELISA reader (MRXII-Dynex). Results and Discussion Pathogenecity of the P.multocida strains. Minimum Lethal Dose as a pathogenecity indicator of P.multocida isolates in mouse, rabbit and chicken is presented in table 1. It was found that P.multocida strain PMI030 (serotype 1) was highly virulent in all tested animals. Injection of P.multocida strains (with different MLD) induced lethality in rabbit. Strain PMI030 was found to be highly virulent for chicken. Injection of only 20CFU killed all birds in challenged group whereas, inoculation of CFU of PMI035 killed all inoculated birds. None of the birds injected by CFU of PMI047 died, however all showed some signs of sickness such as anorexia, ruffled feather and diarrhea. P.multocida was isolated from kidney, spleen, heart and bone marrow of all sick and died birds. Table 1. Minimum lethal dose of P.multocida starins in mouse, rabbit and chicken P.multocida strain Mouse Rabbit Chicken PMI PMI PMI Not done* *This strain was low pathogen for chicken Efficacy of the polyvalent vaccine. All chickens challenged with CFU of PM1030 and CFU of PM1047 were survived whereas 7 out of 10 challenged chickens with CFU of PM1035 were survived. Chickens immunized twice with trivalent vaccine were resistant (70-100%) to challenge with homologous serotypes. As serotype 4 (PMI047) P.multocida was a low virulent strain, inoccurance of sickness signs in challenged birds was considered as protective immunity. P.multocida could not be recovered from immunized chickens, which survived the challenge while it could be isolated from all dead or sick birds. The

5 Arch. Razi Ins. 59 (2005) results of homologous and heterologous serotype challenge revaled that effective killed vaccine against fowl cholera should contain the important serotypes to induce broad-spectrum protection. It is well known that the most important serotypes of P.multocida which cause fowl cholera are serotypes 1, 3 and 4 (Glisson et al 2003). Results of measuring the antibody level by ELISA in immunized chickens detected with whole cell and sonicated antigens are shown in table 2. The trivalent fowl cholera vaccine gave significant protection against experimental challenge with each of three serotypes. Table 2. Mean antibody level (optical density) in chickens before vaccination, two and four weeks after vaccination detected by whole cell and sonicated antigens of P.multocida starins Whole cell antigen Sonicated antigen P/N Ratio 4 weeks P/N Ratio 2 weeks Prevaccination Prevaccination P/N Ratio 4 weeks P/N Ratio 2 weeks P.multocida strain PMI (0.041) (0.123) (0.149) (0.069) (0.227) (0.136) 3.31 PMI (0.021) (0.121) (0.144) (0.101) (0.215) ( PMI (0.045) (0.101) (0.136) (0.071) (0.127) (0.169) 3.35 The potential use of ELISA test as a practical method for determination of immunological response of poultry to vaccination programs has been evaluated previously (Avakian et al 1989, Sender et al 1989). The method was introduced to detect antibody levels to P.multocida in turkeys (Marshall et al1980), chickens (Dick & Johnson 1984, Hofacre et al 1987) and geese (Kedrak et al 2000). In chickens and turkeys, the antibody titer was measured with ELISA highly correlated

6 108 Jabbari & Moazeni Jula/Arch. Razi Ins. 59 (2005) with protection against challenge with virulent organisms. Results of this study indicated that ELISA test could be valuable in the evaluation of the immune response of vaccinated chickens with killed vaccines after 8 weeks of age. All chickens showed a secondary response greater than that seen in chickens vaccinated once (Table 2). It seems that a significant immunolgical stimulus had been elicited by the second exposure. Such antibody response was well within the expected normal range and would provide protection against a cholera challenge. As antibody levels showed at virulent challenge exposure, it became evident that all birds with P/N ratio of 3.2 or higher survived. So far, whole cell and sonicated antigens of P.multocida have been generally used as coating antigen in ELISA test (Perelman et al 1990, Solano et al 1983). In the present study, the whole cell was used as coating antigen resulted in high sensitivity. Howevere some nonspecific reactions happend when sonicated antigen was used. It seems that when bacterial cells were disrupted by ultrasonication, both internal and external antigenic substances can be solublized, which can be bind nonspecifically to antibodies in the serum. Aknowledgments We thank A.R. Sanchooli, S. Haghighi and B. Malek for their technical assistances. This work has been supported by Ministry of Jahad-e-Agriculture grant No References Avakian, A.P., Dick, J.W. and Derieux, W.T. (1989). Fowl cholera immunity induced by various vaccines in broiler minibreeder chickens determined by enzyme-linked immunosorbent assay. Avian Diseases 33:

7 Arch. Razi Ins. 59 (2005) Avakian, A.P., Dick, J.W. (1989). Antigenic properties of four serotypes of Pasteurella multocida determined by enzyme-linked immunosorbent assay. Avian Diseases 30: Bozorgmehrifard, M.H., Afghan, M. (1971). A case report of fowl cholera. Journal of Faculty of Veterinary Medicine-University of Tehran 28: Glisson, J.R., Hofacre, C.L. and Christensen, J.P. (2003). Fowl cholera. In: Saif, Y.M. (Ed), Diseases of Poultry, (11 th edn). Pp: Iowa State Press. Ames, Iowa. Hofacre, C.L., Glisson, G.R. and Kleven, S.H. (1987). Comparison of vaccination protocols of broiler breeder hens for Pasteurella multocida utilizing enzymelinked immunosorbent assay. Avian Diseases 31: Jabbari, A.R., Vasfi Marandi, M., Saharee, A.A. (2003). Protein fingerprinting of avian Pasteurella multocida and virulence of its prototype in mouse. Journal of the Faculty of Veterinary medicine- Tehran University 58: Jabbari, A.R., Saharee, A.A. and Esmaily, F. (2002a). Characterization of avian Pasteurella multocida isolates by protein profiles and restriction enzyme analysis of chromosomal DNA. Archives of Razi Institute 54:1-15. Jabbari, A.R., Saharee, A.A., Bahaman, A.R., Mutalib, A.R., Ideris, A., Esmaily, F., Vasfi Marandi, M. and Esmailzadeh, M. (2002b). Genetic diversity among avian Pasteurella multocoda isolates by using repetitive element PCR. Iranian Journal Veterinary Research, University of Shiraz 3: Jabbari, A.R., Esmaily, F., Vasfi Marandi, M. and Pourbakhsh, S.A. (2001). Study on biotyping and serotyping of Pasteurella multocida isolated from poultry in Iran. Pajouhesh-va-Sazandegi 52:64-67 (in Persian). Kalaydari, G., Bozorgmehrifard, M.H. and Tabatabaei, A.M. (2004). Isolation and identification of Pasteurella multocida in breeder stocks. Journal of Faculty of Veterinary Medicine-University of Tehran 59:63-65.

8 110 Jabbari & Moazeni Jula/Arch. Razi Ins. 59 (2005) Kedrak, A., Opacka, B.B. and Salamonowicz, E.S. (2000). Use of the ELISA for determination of anti-pasteurella multocida serum level in geese vaccinated against pasteurellosis. Bulletin of theveterinary Institute Pulaway 44: Marshall, M.S., Robinson, R.A. and Jenson, M.M. (1980). Use of an enzyme-linked immunosorbent assay to measure antibody responses in turkeys against Pasteurella multocida. Avian Diseases 25: OIE Standard Commission. (2000). Manual of standard for diagnostic tests and vaccines. OIE Publication. Pp: Perelman, B., Hadash, D., Meroze, M., Gurlavie, A., Abramson, M. and Samberg, Y. (1990). Vaccination of young turkeys against fowl cholera. Avian Pathology 19: Petersen, S.K., Foged., N.T., Bording, A., Neilsen, J.P., Riemann, H.K. and Fradesen, P.L. (1991). Recombinant derivatives of Pasteurella multocida toxin condidates for a vaccine against progresive atrophic rhinitis. Infection and Immunity 59: Rimler, R.B., Rhoades, K.R. (1989). Pasteurella multocida. In: C. Adlam and J.M. Ruther (Eds), Pasteurella and Pasteurellosis. Pp:34-67 and Academic Press, London. Sander, J.E., Resurreccion, R.S., Waltman, W.D. and McMurray, B.L. (1998). Pasteurella challenge and ELISA serology evaluation of broiler breeders vaccinated with live cholera vaccine. Avian Diseases 42: Solano, W., Gimbrone, J.J. and Panangala, V.S. (1983). Comparison of enzymelinked immunosorbent assay and indirect hemagglutination test for quantitating antibody responses in chickens against Pasteurella multocida. Avian Diseases 27: Sotoodehnia, A., Vand Yousefi, J. and Aarabi, I. (1986). Isolation and typing of Pasteurella multocida poultry isolates from Iran. Archives of Razi Institute 36, 37:85-86.

9 Arch. Razi Ins. 59 (2005) Sotoodehnia, A., Aarabi, I., Vand Yousefi, J. and Tavasoli, A. (1984). The efficacy of the autogenous fowl cholera killed aluminium hydroxide vaccine in ducks in Iran. Archives of Razi Institute 34, 35: Tavasoli, A., Sotoodehnia, A., Aarabi, I. and Vand Yousefi, J. (1984). A case report of fowl cholera disease in North of Iran. Archives of Razi Institute 34, 35:39-41.

Comparative immunogenicity of fowl cholera vaccine in Jinding ducks

The Bangladesh Veterinarian (2013) 30(2) : 41-45 Comparative immunogenicity of fowl cholera vaccine in Jinding ducks S Sultana, S Saha and MM Amin* 1 Department of Microbiology and Hygiene, Faculty of